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Technical Specification: Immunohistochemistry Reagents

1. Product Range

Solarbio IHC reagents cover the complete immunohistochemistry workflow — from antigen retrieval and blocking through detection, chromogenic visualization, and counterstaining. All detection systems are validated for formalin-fixed paraffin-embedded (FFPE) tissue sections and frozen sections.

Product SKU Application Format
IHC Detection Kit (HRP/DAB, mouse) SP0020 DAB chromogenic IHC — mouse primary antibodies 50–200 tests
IHC Detection Kit (HRP/DAB, rabbit) SP0022 DAB chromogenic IHC — rabbit primary antibodies 50–200 tests
IHC Detection Kit (HRP/DAB, mouse/rabbit) SP0025 DAB chromogenic IHC — mouse and rabbit primary antibodies 50–200 tests
Antigen Retrieval Buffer (Citrate, pH 6.0) C1030 Heat-induced epitope retrieval (HIER) 500 mL, 1 L (1×)
Antigen Retrieval Buffer (EDTA, pH 9.0) C1032 Heat-induced epitope retrieval (HIER) 500 mL, 1 L (1×)
DAB Chromogen Kit DAB0030 Brown chromogenic detection 500 reactions (15 mL)
Normal Goat Serum S0100 Blocking, IHC/IF 10 mL, 50 mL
Hematoxylin (Mayer's) H8070 Nuclear counterstain 500 mL
Permanent Mounting Medium M8430 Coverslip mounting, xylene-based 15 mL
Aqueous Mounting Medium M8440 Coverslip mounting, water-based 15 mL
IHC PAP Pen P2600 Hydrophobic barrier for tissue sections 1 pen (4 mL)

2. IHC Detection System Specifications

Solarbio IHC Detection Kits use a dextran polymer technology that conjugates multiple HRP molecules to a single polymer backbone, which is already linked to secondary antibodies. This polymer-based detection system provides higher sensitivity (3–5×) than traditional avidin-biotin complex (ABC) methods, with lower background and fewer steps.

Parameter SP0020 / SP0022 / SP0025
Detection mechanism Dextran polymer-HRP conjugated to secondary antibody (polymer chain)
Signal amplification Direct polymer (no biotin-avidin step); ~40 HRP molecules per polymer
Sensitivity Equivalent to or greater than ABC method; no biotin interference
Species reactivity (SP0025) Mouse IgG and rabbit IgG
Primary antibody requirement 100–500 μg/mL for optimal signal
DAB incubation 3–10 min at RT (monitor microscopically)
Counterstain Hematoxylin (Mayer's), 30–60 s
Total detection time Approximately 30 min (after primary antibody)
Storage 2–8°C, 12 months (do not freeze)
Endogenous biotin interference Eliminated (no biotin in detection system)
Endogenous peroxidase Requires 3% H₂O₂ blocking step (included in kit)

3. Antigen Retrieval Buffer Specifications

3.1 Mechanism of Antigen Retrieval

Formalin fixation creates methylene cross-links between proteins, masking epitopes and reducing antibody accessibility. Heat-induced epitope retrieval (HIER) uses high temperature (95–120°C) to hydrolyze these cross-links, restoring protein conformation for antibody binding. The pH of the retrieval buffer critically affects which epitopes are exposed.

3.2 Citrate Buffer (pH 6.0, C1030) vs. EDTA Buffer (pH 9.0, C1032)

Parameter Citrate Buffer (pH 6.0) EDTA Buffer (pH 9.0)
Composition 10 mM sodium citrate, 0.05% Tween 20 1 mM EDTA, 0.05% Tween 20
Final pH (1×) 6.0 ± 0.1 9.0 ± 0.1
Retrieval mechanism High-temperature hydrolysis at mild pH High-temperature hydrolysis at alkaline pH; stronger cross-link breakage
Recommended antibodies Nuclear and cytoplasmic targets Membrane-bound and some nuclear targets
Tissue compatibility All tissue types All tissue types; may cause section detachment with prolonged heating
Heating method Microwave, autoclave, pressure cooker, water bath Microwave, autoclave, pressure cooker, water bath
Typical heating time 15–20 min at 95–100°C 10–15 min at 95–100°C
Cooling time 20–30 min at RT 20–30 min at RT
Reuse Single use only Single use only

3.3 Antigen Retrieval Decision Guide

Target Recommended Buffer Notes
Ki-67 (nuclear proliferation marker) Citrate pH 6.0 20 min microwave; excellent nuclear signal
HER2/neu (membrane, breast cancer) EDTA pH 9.0 Alkaline retrieval enhances membrane staining
CD3 (T-cell membrane) EDTA pH 9.0 Membrane signal stronger with alkaline retrieval
p53 (nuclear, tumor suppressor) Citrate pH 6.0 Nuclear accumulation detectable
CK7/CK20 (cytoplasmic, cytokeratins) Citrate pH 6.0 or EDTA pH 9.0 Both work; citrate preferred for cleaner background
ER/PR (nuclear, hormone receptors) Citrate pH 6.0 Standard for breast cancer IHC panels
CD20 (B-cell membrane) Citrate pH 6.0 Robust membrane staining
PD-L1 (membrane/cytoplasmic) EDTA pH 9.0 Alkaline retrieval improves PD-L1 staining
Vimentin (cytoplasmic) EDTA pH 9.0 Strong signal with alkaline buffer

4. DAB Chromogen Kit (DAB0030)

Principle: 3,3′-Diaminobenzidine (DAB) is oxidized by HRP in the presence of hydrogen peroxide to produce a brown, insoluble precipitate at the site of the antigen-antibody reaction. The DAB polymer is alcohol- and xylene-resistant, allowing for permanent mounting.

DAB (reduced, colorless)  →  DAB (oxidized, brown precipitate)
                    ↑     
                HRP + H₂O₂
Parameter Specification
Composition DAB tablet (10 mg) + DAB buffer (15 mL H₂O₂ in stabilizing solution)
Preparation Dissolve 1 DAB tablet in 1 mL DAB buffer; then add 14 mL distilled water
Working solution stability 2–8°C, 1 week (dark)
Color Brown
Water solubility (after oxidation) Insoluble (permanent)
Counterstain compatibility Hematoxylin (blue nuclei); methyl green
Dehydration Dehydrate through graded alcohols, clear in xylene
Mounting Xylene-based permanent mounting medium
Sensitivity 3–5 ng of target per section (approximately 100–500 molecules/cell)
Storage -20°C, 12 months (tablet desiccated)

5. Protocol Overview

Step Reagent Time Temperature Notes
1. Deparaffinize/Hydrate Xylene (3×), 100% → 70% ethanol, water 30 min RT Complete deparaffinization is critical; insufficient xylene time → patchy staining
2. Antigen retrieval Citrate pH 6.0 or EDTA pH 9.0 15–20 min 95–100°C Microwave 5 min × 3 at medium-high power; do not boil dry
3. Cool Retrieval buffer 20–30 min RT Let slides cool in buffer; rapid cooling may cause antigen collapse
4. Block endogenous peroxidase 3% H₂O₂ in methanol or PBS 10–15 min RT Covers entire section; wash 3× PBS
5. Blocking Normal goat serum (10% in PBS) 30 min RT Do not wash; tap off excess
6. Primary antibody Diluted in PBS Overnight 4°C Or 1 h at 37°C; use humidified chamber
7. Wash PBS 5 min × 3 RT Gentle agitation
8. Detection system HRP-polymer conjugate 30 min RT Eliminates need for biotin blocking
9. Wash PBS 5 min × 3 RT Thorough washing reduces background
10. DAB DAB + H₂O₂ substrate 3–10 min RT Monitor microscopically; stop when target turns brown
11. Wash Distilled water 5 min RT Remove excess DAB
12. Counterstain Hematoxylin (Mayer's) 30–60 s RT Filter hematoxylin before use
13. Blue Tap water (bluing) 5 min RT Change water until clear
14. Dehydrate 70% → 100% ethanol, xylene 5 min each RT Clear in xylene before mounting
15. Mount Permanent mounting medium — RT Coverslip; avoid air bubbles

6. Troubleshooting

Issue Possible Cause Solution
No staining Antigen retrieval failed; primary antibody inactive Increase retrieval time; verify primary on positive control tissue
Weak staining Insufficient retrieval; primary dilution too low Titrate primary; use EDTA pH 9.0 instead of citrate; extend DAB to 10 min
High background (nuclear) Primary antibody cross-reactivity; retrieval too harsh Reduce retrieval time; try reduced primary concentration
High background (diffuse) Inadequate blocking; secondary polymer overspill Increase blocking to 1 h; wash more thoroughly
Tissue detachment Over-heating during retrieval; slides not coated Use poly-L-lysine or silane-coated slides; reduce heating time
DAB precipitates in solution DAB solution old; self-polymerization Prepare fresh DAB; use within 15 min
Patchy staining Incomplete deparaffinization; uneven antigen retrieval Extend xylene time to 30 min; ensure full coverage with retrieval buffer
Hematoxylin too dark Over-counterstaining Reduce hematoxylin to 15–20 s; use automated staining

7. Cross-References

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